Phosphorylation is an important signaling mechanism that occurs through post-translational modification of proteins. Kinases are enzymes that add a phosphate group to serine, threonine, tyrosine, histidine, arginine, and lysine amino acid residues of their substrate proteins.
Phosphorylation can have several effects including activation, inactivation, structural changes, binding to other proteins, and protein degradation. Kinase upregulation and activation is common in many diseases including cancers. In fact, the first oncogene identified was Src, a tyrosine kinase which drives cancer by increasing phosphorylation and signaling through downstream substrates.
Therefore, phosphorylated proteins or phosphoproteins are important biomarkers and their detection is critical in the study, diagnosis, and treatment of disease. Furthermore, cell-to-cell heterogeneity in cancers has recently been identified as an important factor in drug resistance. Therefore, there is a need for single-cell and single-molecule analysis of phosphoproteins.
Existing methods to detect phosphorylated proteins include antibody-based methods such as immunofluorescence, enzyme-linked immunosorbent assay (ELISA), flow cytometry, Western blotting, radiometric kinase assays, and mass spectrometry. Kinase assays are generally performed in vitro and require the use of purified kinase. Thus, kinase assays are not a useful technique for detecting changes in substrate phosphorylation under different in vivo conditions. Immunofluorescence can detect cell-to-cell heterogeneity, however sensitivity and antibody performance are frequent issues. Further, immunofluorescence and Western blotting are generally qualitative techniques.
ELISA can be quantitative, but the volumes required for analysis do not allow for single-cell studies. Flow cytometry does allow for single-cell studies and quantification of the number of cells labeled, but does not allow for single-molecule detection and quantification within each cell. Additionally, flow cytometry of internal phosphoproteins requires fixation and permeabilization of cells and subsequent recovery of proteins is not possible. Finally, mass spectrometry can be sensitive and qualitative, but due to the negative charge of phosphate groups, phosphorylated proteins are often difficult to ionize and detect over background peaks.
Therefore, a more robust and quantitative method for the detection and collection of phosphoproteins is needed.